Two-Step Activity-Based Protein Profiling with the Proteasome System as Model of Study

Area of Science:

• Biochemistry
• Chemical Biology
• Proteomics

Background:

• Activity-based protein profiling (ABPP) identifies enzyme activities using chemical probes that target catalytic nucleophiles.
• Traditional ABPP probes often have reporter tags, which can interfere with enzyme activity or probe delivery.
• A ligation handle allows for post-reaction attachment of reporters, offering temporal and spatial control.

Purpose of the Study:

• To demonstrate a triple bioorthogonal ligation strategy for simultaneous profiling of multiple proteasome activities.
• To circumvent disadvantages of pre-attached reporter tags in chemical probes for ABPP.
• To validate the efficacy of sequential bioorthogonal ligations within a single experimental workflow.

Main Methods:

• Utilized a norbornene-tagged probe selective for the proteasome β5-subunit, ligated with fluorescent tetrazine.
• Employed an azide-functionalized probe selective for the β1-subunit, reacted with biotinylated phosphine.
• Applied an alkyne-substituted pan-reactive probe for β2 activity, followed by ligation with an azide-coupled fluorophore.

Main Results:

• Successfully demonstrated a triple bioorthogonal ligation strategy for proteasome activity profiling.
• The combined triple ligation yielded results comparable to individual ligation reactions.
• Validated the utility of sequential bioorthogonal ligations for complex proteomic analysis in a single experiment.

Conclusions:

• The triple bioorthogonal ligation strategy is a powerful tool for simultaneous ABPP of multiple enzyme activities.
• This approach offers enhanced control over reporter tag attachment, improving ABPP workflows.
• The method is effective for comprehensive analysis of proteasome enzymatic activities.